KR20220105801A - Biosensor for Alkaline phosphatase measurement - Google Patents

Abstract

The present invention relates to a biosensor for measuring ALP which can quantify ALP in a biological sample through electrochemical analysis and thus, has an effect of performing a liver function test simply and quickly. The biosensor and an external device are electrically connected through a USB or a pin-type connection terminal such that costs can be reduced by being applicable to various devices and the convenience and usability of a product can be increased by being easily used in real life. In addition, the biosensor of the present invention comprises: a connection module electrically connected to the external device; and a sensing module detachable from the connection module such that various types of inspections can be performed by replacing the sensing module and only the sensing module can be replaced in a state where the connection module is coupled to the external device to perform an inspection continuously and rapidly.

Description

Biosensor for ALP measurement {Biosensor for Alkaline phosphatase measurement}

The present invention relates to a biosensor for measuring ALP.

Alkaline phosphatase (ALP) is an enzyme found in tissues throughout the body, such as the liver, bones, kidneys, intestines, and placenta of pregnant women. However, the concentration of alkaline phosphatase is highest in the cells of the bone and liver. In the liver, it is found at the edge of the cells that form the bile ducts, which are small ducts that drain bile from the liver into the intestines and aid in the digestion of fats in the diet. Bone alkaline phosphatase is produced by specific cells called 'osteoblasts' that are involved in bone formation. Each different tissue produces a characteristic form of alkaline phosphatase called isoenzyme.

Elevated blood alkaline phosphatase concentrations are most commonly observed with liver disease or bone lesions. The increase in enzyme concentration can be greatest when the bile duct is blocked. People with liver cancer, cirrhosis, taking drugs that are toxic to the liver, and hepatitis can also increase the level of enzymes in the blood, although not as severe as they are. Alkaline phosphatase levels can be increased in any event that promotes excessive bone formation, such as Paget's disease, rheumatoid arthritis, or the timing of fracture healing. Accordingly, liver function and the like can be diagnosed by measuring the level of alkaline phosphatase.

On the other hand, target substances such as hormones, proteins, and pathogens, which enable prediction of human disease symptoms and progression, and further the condition of food, can be analyzed by immunoassay using antigen-antibody reaction. On the other hand, analysis of target substances based on immunoassay has been usually performed in clinical laboratories equipped with special equipment. However, as the need for self-diagnosis at home and for examinations at medical sites in hospitals or emergency rooms has recently increased, the development of an immunoassay platform that does not require specialized knowledge or complicated processes and has a short analysis time has been continuously required. .

Therefore, as a method for this, there is a need to develop a biosensor capable of fast and accurate analysis of alkaline phosphatase by electrochemical analysis.

One aspect is a connection module electrically connectable to an external device; A sensing module configured to detect an Alkaline phosphatase (ALP) material from the analyte sample introduced into the interior, and transmit an electrical signal generated by reacting with the detected ALP material to the connection module; Containing, the sensing module includes: , A sensor for detecting the ALP material from the analyte sample and reacting with the ALP material to generate the electrical signal is provided, the ALP material is quantitatively measured by the generated electrical signal, the sensor, A substrate; and a plurality of electrodes provided on one surface of the substrate to react with the ALP material to generate an electrochemical signal, wherein the sensing module is detachable from the connection module.

One aspect is a connection module electrically connectable to an external device; A sensing module configured to detect an Alkaline phosphatase (ALP) material from the analyte sample introduced into the interior, and transmit an electrical signal generated by reacting with the detected ALP material to the connection module; Containing, the sensing module includes: , A sensor for detecting the ALP material from the analyte sample and reacting with the ALP material to generate the electrical signal is provided, the ALP material is quantitatively measured by the generated electrical signal, the sensor, and a plurality of electrodes provided on one surface of the substrate and reacting with the ALP material to generate an electrochemical signal, wherein the sensing module is detachable from the connection module.

In one embodiment, the sensor may include a nanowell, microwell, or milliwell structure including a plurality of grooves on the electrode.

In one embodiment, the nanowell, microwell, or milliwell may have a diameter of 50 nm to 50 mm.

In one embodiment, the analysis sample may be a biological sample isolated from a subject.

In one embodiment, the electrode may be a working electrode, a counter electrode, or a reference electrode.

In one embodiment, the connection module and the sensing module may be detachably coupled to each other through magnetism, and may be electrically connected to each other through surface contact.

In one embodiment, the sensing module may be one or more, and the one or more sensing modules may be detachable from one connection module.

In one embodiment, the quantitative measurement of the ALP substance may be measured by any one of Equations 1 to 3:

[Equation 1]

y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL))

[Equation 2]

y(uA) = -1.35 + 1.15*exp(-1.74*107x(g/mL))

[Equation 3]

y(uA) = -2.76 + 1.63*exp(-3.53*107x(g/mL))

The present invention relates to a biosensor for measuring ALP, and it is possible to quantify ALP in a biological sample through electrochemical analysis, thereby having the effect of performing a liver function test simply and quickly. Since it is configured to electrically connect the biosensor and an external device through a USB or pin-type connection terminal, it can be applied to a variety of devices to reduce costs, and it can be easily used in real life for convenience and usability of the product. There are advantages to which this can be increased.

In addition, since the biosensor of the present invention is composed of a connection module electrically connected to an external device and a sensing module detachable from the connection module, various types of tests can be performed by replacing the sensing module, and the connection module is an external device. Since only the sensing module can be replaced in the state of being coupled to the

1 is a state diagram showing a state in which a biosensor according to an embodiment of the present invention is connected to an external device.
2 is a perspective view illustrating a biosensor in which a milliwell or microwell structure is formed according to an embodiment of the present invention.
3 is a plan view illustrating a disassembled state of a biosensor according to an embodiment of the present invention.
FIG. 4 is a cross-sectional view taken along line VI-VI of FIG. 4 .
FIG. 5 is a side view showing part “A” of FIG. 4 .
6 is a plan view schematically illustrating a disassembled state of a biosensor according to another embodiment of the present invention.
FIG. 7 is an enlarged view of part “B” of FIG. 6 .
8 is a plan view schematically illustrating a state in which a biosensor according to another embodiment of the present invention is disassembled.
FIG. 9 is a view schematically illustrating a process in which part “C” of FIG. 8 is coupled.
10 is a side view schematically illustrating a state in which a biosensor according to another embodiment of the present invention is disassembled.
11 is a graph showing current values measured for each concentration of ALP using a nanowell electrode.
12 is a graph showing current values measured for each concentration of ALP using a microwell electrode.
13 is a graph showing current values measured for each concentration of ALP using a milliwell electrode.
14 is a graph showing the conversion of a current value measured for each ALP concentration by a ratio divided by a negative control of each electrode in order to compare the sensitivity of each electrode.
15 is a graph showing the relationship between the current value and the ALP concentration in the nanowell electrode.
16 is a graph showing a relationship between a current value and an ALP concentration in a microwell electrode.
17 is a graph illustrating a relationship between a current value and an ALP concentration in a milliwell electrode.

This specification clarifies the scope of the present invention, explains the principles of the present invention, and discloses embodiments so that those of ordinary skill in the art to which the present invention pertains can practice the present invention. The disclosed embodiments may be implemented in various forms.

Expressions such as “comprises” or “may include” that may be used in various embodiments of the present invention indicate the existence of the disclosed function, operation, or component, and may include one or more additional functions, operations, or components, etc. are not limited. In addition, in various embodiments of the present invention, terms such as "comprise" or "have" are intended to designate that a feature, number, step, action, component, part, or combination thereof described in the specification is present, It should be understood that it does not preclude the possibility of addition or existence of one or more other features or numbers, steps, operations, components, parts, or combinations thereof.

Terms including an ordinal number, such as first, second, etc., may be used to describe various elements, but these elements are not limited by the above-described terms. The above terminology is used only for the purpose of distinguishing one component from another component.

When a component is referred to as being “connected to and coupled to” another component, the component may be directly connected or coupled to the other component, but between the component and the other component. It should be understood that there may be other new components in the On the other hand, when it is said that an element is "directly connected" or "directly coupled" to another element, it will be understood that no new element exists between the element and the other element. should be able to

Meanwhile, as used herein, a “module” or “unit” for a component performs at least one function or operation. In addition, a “module” or “unit” may perform a function or operation by hardware, software, or a combination of hardware and software. In addition, a plurality of “modules” or a plurality of “units” other than a “module” or “unit” that must be performed in specific hardware or are executed in at least one processor may be integrated into at least one module. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be abbreviated or omitted.

The present invention relates to a biosensor for ALP measurement, and to a biosensor in which a sensing part can be replaced by using a connection module electrically connectable to an external device and a sensing module detachable from the connection module. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Referring to FIG. 1 , the biosensor 10 according to an embodiment of the present invention uses a nanowell, microwell, or milliwell structure 220 to improve resolution, and transmits an electrical signal generated from the biosensor 10 . It is configured to be electrically connected to the external device 20 in which the software capable of measuring and analyzing is installed.

Specifically, the biosensor 10 is electrically connected to the external device 20 in the form of a smartphone or tablet as shown in FIG. can be electrically connected. However, the external device 20 is not limited thereto, and may be used in various forms electrically connectable to the biosensor 10 .

The external device 20 has a terminal unit (not shown) that can be coupled to the first connection terminal 110 provided in the connection module 100 so as to be electrically connectable to the connection module 100 of the biosensor 10 to be described later. may be provided. In addition, when the external device 20 is connected to the biosensor 10, a program capable of performing qualitative and quantitative analysis of the ALP material by measuring and analyzing the potential according to the redox reaction of the biosensor 10 or Applications and the like may be installed. Through this, the external device 20 collects and analyzes signals generated by the biosensor 10, and can detect various diseases or diseases, or measure and display changes thereof.

In addition, the external device 20 may be connected to the Internet through wired or wireless communication and transmit data measured through the biosensor 10 to a user terminal or a server of a medical institution. Through the transmitted data, the subject or medical institution can monitor the health status in real time.

For example, for example, the external device 20 includes an input unit (not shown) electrically connected to the biosensor 10 to which the biosensor 10 is coupled, a signal (data) transmitted through the input unit, A control unit for analysis and diagnosis (not shown), a display unit for outputting data transmitted from the control unit in an externally identifiable form (not shown), and a communication unit for transmitting data received from the control unit to a selected user terminal and a medical institution server (not shown) ) and a power supply unit (not shown) for supplying power to an input unit, a control unit, a display unit, and a communication unit.

The biosensor according to an embodiment of the present invention includes a connection module 100 , a sensing module 200 , and a sensor 210 . Hereinafter, preferred embodiments of the present invention will be described in detail.

2 and 3 , the connection module 100 is electrically connectable to the external device 20 . To this end, the connection module 100 includes a first connection terminal 110 , a second connection terminal 120 , a controller 130 , and a body 140 .

The first connection terminal 110 is coupled to the external device 20 to be electrically connectable to the external device 20 . The first connection terminal 110 may have various configurations as long as it can be electrically connected to the external device 20 .

The first connection terminal 110 is coupled to the terminal provided in the external device 20 in a shape corresponding to the terminal of the external device 20 so as to be electrically connected to the external device 20 . can be done Specifically, the first connection terminal 110 may be provided in the form of a USB, micro USB, or pin (PIN), but is not limited thereto, and various configurations are possible if it can be electrically connected to the external device 20 . can be used

The second connection terminal 120 is coupled to the sensing module 200 to be described later and is electrically connectable to the sensing module 200 . The second connection terminal 120 may be provided in the form of a micro USB or pin, but is not limited thereto, and various configurations may be used as long as it can be electrically connected to the sensing module 200 .

The controller 130 is electrically connected to the first connection terminal 110 and the second connection terminal 120, and a signal ( data) may be configured to control the transmission. The controller 130 may be electrically connected to the first connection terminal 110 and the second connection terminal 120 through an electric wire, but is not limited thereto. If the two connection terminals 120 can be electrically connected, various configurations may be used.

The main body 140 includes a lower body in which the first connection terminal 110 , the second connection terminal 120 , and the controller 130 are installed, and is coupled to the lower body to provide the first connection terminal 110 . , the second connection terminal 120 , and an upper body protecting an upper portion of the controller 130 may be included.

Referring to FIGS. 2 and 3 , the sensing module 200 detects an ALP material from an analysis sample introduced therein, and transmits an electrical signal generated by reacting with the detected ALP material to the connection module 100 . A nanowell, microwell, or milliwell structure 220 is provided while being configured to transmit.

The analysis sample may refer to a biological sample isolated from a subject, and may be blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, intestinal fluid, genitourinary tract fluid. , breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof. However, the analysis sample is not limited thereto, and may be a variety of materials as long as it is a biological sample. In addition, the biological sample may include an intact protein. The intact protein may be a protein isolated from a biological sample without further modification of the protein. An intact protein may be a protein that has been isolated from a biological sample, for example, without proteolysis by proteolytic enzymes.

The sensing module 200 may include a sensor 210 that detects the ALP material from the analyte sample, reacts with the ALP material to generate the electrical signal, and the sensor 210 includes a substrate 211 , a plurality of electrodes 212 , and a nanowell, microwell or milliwell structure 220 .

Specifically, the sensor 210 may be provided with a sensing material for detecting the ALP material by reacting with the ALP material contained in the analyte sample. When the sensor 210 comes into contact with the analyte sample, it interacts with the ALP material included in the analyte sample to generate an electrical signal. The ALP material may be quantitatively measured by the generated electrical signal. Quantitative measurement of the ALP material may be measured by any one of Equations 1 to 3 below.

[Equation 1]

y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL))

[Equation 2]

y(uA) = -1.35 + 1.15*exp(-1.74*107x(g/mL))

[Equation 3]

y(uA) = -2.76 + 1.63*exp(-3.53*107x(g/mL))

Accordingly, the external device 20 connected to the biosensor 10 can analyze the electrical signal generated from the biosensor 10 to detect the presence or concentration of the ALP material, and analyze only the electrical signal. Because the presence or concentration of ALP can be quickly detected, diseases such as liver function can be quickly and easily diagnosed. However, the sensor 210 is not limited thereto, and may be configured to move, stop, filter, purify, react, and mix the analysis sample.

The substrate 211 provided in the sensor 210 may have a predetermined size, and the substrate 211 may have a size of 2 x 2 mm. However, the size of the substrate 211 is not necessarily limited thereto, and may be changed to various sizes.

In addition, the substrate 211 may be made of a material that has elasticity and is bendable. For example, the substrate 211 may be a polyurethane (Poly urethane, PU)-based, polydimethylsiloxane (PDMS)-based, NOA (Noland Optical Adhesive)-based, epoxy-based, polyethylene terephthalate, At least one of PET), polymethyl methacrylate (PMMA), polyimide (PI), polystyrene (PS), polyethylene naphthalate (PEN), and polycarbonate (PC) It may be made of a material of

However, the material of the substrate 211 is not necessarily limited thereto, and electrodes generating a potential difference according to a reaction with a target material may be disposed, and various materials having flexibility may be applied.

The electrode 212 may be provided in plurality, and the electrode 212 may be provided on one surface of the substrate 211 and may react with a target material included in the analysis sample to generate an electrochemical signal. .

The electrode may be a working electrode, a counter electrode, or a reference electrode.

The plurality of electrodes 212 includes a first electrode 212a reacting with the ALP material for oxidation or reduction, a second electrode 212b for oxidation or reduction reaction with the ALP material in opposition to the reaction of the first electrode 212a, and a third electrode 212c configured to maintain a constant operating voltage between the first electrode 212a and the second electrode 212b.

The plurality of electrodes 212 may be formed on the insulating substrate 211 through a screen printing method. The second electrode 212b and the third electrode 212c may be disposed to surround the first electrode 212a.

The first electrode 212a and the second electrode 212b may be disposed on the substrate 211 to be spaced apart from each other by a predetermined distance. The first electrode 212a may have a circular shape, and the second electrode 212b may have a semicircular shape to surround a portion of the first electrode 212a. However, the arrangement shape of the first electrode 212a and the second electrode 212b and the shape of each of these electrodes are not limited thereto, and may be changed as necessary.

The first electrode 212a may refer to a working electrode that performs oxidation or reduction reaction by reaction with an ALP material or an analysis sample including an ALP material on the substrate 211 .

A bioreceptor (not shown) specifically binding to the ALP material may be disposed on the first electrode 212a. A pillar having a conductive layer deposited thereon is disposed on the first electrode 212a, and a bioreceptor such as an antibody, antigen, or aptamer may be disposed on the pillar. A material that reacts with the ALP material to induce an electrochemical signal may be further disposed on the first electrode 212a.

Through the pillars and bioreceptors disposed on the first electrode 212a and the second electrode 212b, the reaction area with the ALP material is widened, and through this, the biosensor 10 absorbs a small amount of the ALP material. It can also provide sensitive qualitative and quantitative analysis results. Here, the pillar may be a polymer structure having a nano size.

The pillar may be made of at least one of polyurethane, polydimethylsiloxane, Norland Optical Adhesives (NOA), epoxy, polyethylene terephthalate, polymethyl methacrylate, polyimide, polystyrene, polyethylene naphtharate, polycarbonate, and combinations thereof. have.

The pillars may also be made of a combination of polyurethane and NOA (eg NOA 68). However, the pillars are not limited thereto, and may be made of more various polymers as long as they have flexibility.

In addition, the conductive layer deposited on the pillar may refer to a layer made of a conductive material. The conductive layer may be formed of at least one of Ni, Zn, Pd, Ag, Cd, Pt, Ga, In, and Au, but is not limited thereto.

The second electrode 212b may refer to a counter electrode facing the first electrode 212a on the substrate 211 . Accordingly, if an oxidation reaction occurs in the first electrode 212a by reaction with the ALP material, a reduction reaction may occur in the second electrode 212b. The above-described pillars having a conductive layer deposited thereon may be disposed on the second electrode 212b.

The third electrode 212c may refer to a reference electrode whose potential is stably maintained even when in contact with the ALP material. A reaction layer capable of maintaining a constant potential even when in contact with the ALP material may be provided on the third electrode 212c.

The reaction layer is Ag/AgCl, Ag, Hg2SO4, Ag/Ag+, Hg/Hg2SO4, RE-6H, Hg/HgO, Hg/Hg2Cl2, Ag/Ag2SO4, Cu/CuSO4, KCl saturated calomel half cell (SCE) and salt bridge The potential difference of platinum may be made of a known material in advance.

Referring to FIG. 2 , the nanowell, microwell, or milliwell structure 220 is provided on the electrode 212 and includes a plurality of grooves. The nanowell, microwell or milliwell structure 220 may be provided at an inlet into which the analysis sample is inserted into the sensing module 200 , and the nanowell, microwell or milliwell structure 220 is There is an advantage that the resolution can be improved as it is used.

Specifically, the nanowell, microwell or milliwell structure 220 may be provided on a working electrode, and the nanowell, microwell or milliwell structure 220 is the working electrode. It may be provided on the first electrode 212a.

In addition, the nanowell, microwell, or milliwell may have a diameter of 50 nm to 50 mm. Specifically, 50 nm to 30 mm, 50 nm to 10 mm, 50 nm to 900 μm, 50 nm to 600 μm, 50 nm to 300 μm, 50 nm to 100 μm, 100 nm to 50 mm, 100 nm to 30 mm , 100 nm to 10 mm, 100 nm to 900 μm, 100 nm to 600 μm, 100 nm to 300 μm, 300 nm to 50 mm, 300 nm to 30 mm, 300 nm to 10 mm, 300 nm to 900 μm, 300 nm to 600 μm, 500 nm to 50 mm, 500 nm to 30 mm, 500 nm to 10 mm, 500 nm to 900 μm, 700 nm to 50 mm, 700 nm to 30 mm 700 nm to 10 mm 700 nm to 900 μm can be

The sensing module 200 according to an embodiment of the present invention may further include a connection pad 230 , a connection port 240 , a housing 250 , and a bonding wire 260 .

A single or a plurality of connection pads 230 may be provided, and may be electrically connected to the sensor 210 through the bonding wire 260 . 3 and 5 , on the lower surface of the sensor 210 , a plurality of connection pads 230 and a plurality of connection pads 213 electrically connected to the plurality of electrodes 212 provided in the sensor 210 are connected to each other. ) may be further provided.

One surface of the connection pad 213 attached to the bottom surface of the sensor 210 is electrically connected to the plurality of electrodes 212 provided in the sensor 210 , and the other surface of the connection pad 213 is the It may be electrically connected to the plurality of connection pads 230 through the bonding wire 260 . Specifically, the plurality of connection pads 230 and the connection pads 213 may be provided in a copper foil shape, and a surface of the plurality of connection pads 230 may be further coated with gold foil.

In addition, the connection pad 213 and the sensor 210 may be electrically connected to each other through a conductive material (not shown), and a double-sided adhesive carbon tape may be used as the conductive material. However, the conductive material is not limited thereto, and various materials may be used as long as the connection pad 213 and the sensor 210 can be electrically connected.

The bonding wire 260 is made of gold (Au), and one end of the bonding wire 260 is bonded to the connection pad 213 attached to the sensor 210 , and is provided in the sensor 210 . The electrodes 212 may be electrically connected, and the other end of the bonding wire 260 may be bonded to the plurality of connection pads 230 to be electrically connected to the plurality of connection pads 230 .

2 and 3 , the connection port 240 may be electrically connected to the plurality of connection pads 230 , and may be coupled to the connection module 100 to be electrically connected to the connection module 100 . there will be

The connection port 240 may be electrically connected to the plurality of connection pads 230 through an electric wire 261 , and when coupled to the connection module 100 , the sensor ( Power may be supplied to the 210 , and an electrical signal generated from the sensor 210 may be transmitted to the connection module 100 . The connection port 240 may be provided in a shape corresponding to the second connection terminal 120 provided in the connection module 100 .

The housing 250 includes the sensor 210 , the plurality of connection pads 230 , and the connection port 240 installed therein. The housing 250 includes the sensor 210 and the plurality of connection pads ( 230), may be configured to surround the circumference of the connection port 240 to protect the sensor 210, the plurality of connection pads 230, and the connection port 240 from the outside.

The housing 250 is disposed opposite to the first member 251 and the first member 251 having a channel 253 for guiding the analyte sample provided from the outside to the electrode 212 of the sensor 210 . and is coupled to the first member 251 and includes a second member 252 on which the sensor 210 , the plurality of connection pads 230 , and the connection port 240 are seated.

The channel 253 is a hole formed through the first member 251 , and the channel 253 is an inclined surface 254 for guiding an analysis sample provided from the outside to the electrode 212 of the sensor 210 . ) is included. The inclined surface 254 may be provided along the circumference of the channel 253 , and the analysis sample provided from the outside moves along the inclined surface 254 stably to the electrode 212 of the sensor 210 . be able to reach

Here, referring to FIG. 4 , the nanowell, microwell, or milliwell structure 220 may be provided in the channel 253 . The nanowell, microwell, or milliwell structure 220 may be provided on the first electrode 212a-working electrode, and the channel 253 may be formed on the first electrode 212a. - Can be provided on top of the working electrode (working electrode).

Accordingly, the nanowell, microwell, or milliwell structure 220 is preferably provided in the channel 253 , and the nanowell, microwell or milliwell structure 220 is coupled to the channel 253 . can be supported. By providing the nanowell, microwell or milliwell structure 220 in the channel 253 , the resolution can be improved when the analyzed sample is moved to the electrode 212 through the channel 253 . do.

The first member 251 may be coupled to the second member 252 to cover the sensor 210 and the connection port 240 disposed on the second member 252 to protect them from the outside. . Also, the first member 251 may press the sensor 210 and the connection port 240 with a predetermined force to firmly support them. In addition, the first member 251 prevents the analysis sample provided to the sensor 210 through the channel 253 from leaking to the outside of the housing 250 through coupling with the second member 252 . can do.

3 and 5 , the sensor 210 , the plurality of connection pads 230 , and the connection port 240 are accommodated between the first member 251 and the second member 252 . and an accommodation space 250a for protecting the bonding wire 260 may be provided. The accommodating space 250a may be provided in the form of an intaglio pattern inside the first member 251 and the second member 252, respectively.

Referring to FIG. 4 , at least one coupling protrusion 252a coupled to the first member 251 and supporting the first member 251 is provided on the second member 252 , One member 251 may be provided with at least one coupling groove 251a corresponding to the coupling protrusion 252a.

When the coupling protrusion 252a and the coupling groove 251a are coupled, the center of the channel 253 and the center of the sensor 210 may be naturally aligned at a predetermined position. On the inside of the coupling groove 251a, when the coupling protrusion 252a is inserted into the coupling groove 251a, a sealer made of a rubber or silicone material that seals the space between the coupling protrusion 252a and the coupling groove 251a. may be further provided.

4, the second member 252 has a receiving groove 252b in which the sensor 210 is seated and supported, and a collection groove 252c in which the analysis sample flowing down from the sensor 210 is stored. This may be further provided. A sample guide surface 252d for guiding the analysis sample flowing out of the sensor 210 toward the collection groove 252c may be further provided between the sensor 210 and the collection groove 252c, and the sample guide surface (252d) may be formed in an inclined shape.

Referring to FIGS. 2 and 3 , the housing 250 includes a flow state of an analyte sample, a leak of an analyte sample, an alignment state of the channel 253 and the sensor 210 , and the bonding wire 260 through The sensor 210 and the plurality of connection pads 230 are connected to each other and the electrical connection of the plurality of connection pads 230 and the connection port 240 can be easily observed from the outside. can be

For example, the housing 250 is made of at least one of polymethyl methacrylate, polycarbonate, cyclic olefin copolymer, polyethylene sulfone, and polystyrene, or at least two or more of them. It may be provided with these combined materials. However, the material of the housing 250 is not necessarily limited thereto, and may be made of a polydimethylsiloxane material, which is a silicone-based organic polymer.

The biosensor using a nanowell, microwell, or milliwell structure according to an embodiment of the present invention may be modified and used as follows.

Referring to FIG. 6 , a plurality of sensing modules 200 of the biosensor according to an embodiment of the present invention may be provided, and the plurality of sensing modules 200 are detachably coupled to the connection module 100 . can be That is, the connection module 100 may be electrically connected to the plurality of sensing modules 200 .

To this end, the connection module 100 includes the first connection terminal 110 that can be coupled to the external device 20 and a second connection terminal 120 that can be coupled to the sensing module 200, A plurality of 2 connection terminals 120 may be provided. The plurality of sensing modules 200 may be selectively coupled to the plurality of second connection terminals 120 .

As described above, when a plurality of the sensing modules 200 are provided and a plurality of the second connection terminals 120 are provided, there is an advantage in that it is possible to detect ALP materials of different entities and perform a plurality of tests at once. .

Here, when the plurality of sensing modules 200 are all connected to the plurality of second connection terminals 120 to generate electrical signals, the controller 130 is configured to operate through the plurality of second connection terminals 120 . After storing the order in which the electrical signals are transmitted, the electrical signals of the sensing module 200 connected to the corresponding second connection terminal 120 may be sequentially received and transmitted to the first connection terminal 110 .

The connection module 100 is a power supply unit 150 for controlling the connection between the plurality of second connection terminals 120 and the controller 130 between the plurality of second connection terminals 120 and the controller 130 . may further include.

A plurality of the power supply unit 150 may be provided, and may be individually connected to the plurality of second connection terminals 120 . Also, the power supply unit 150 may be connected to each of the controllers 130 .

The power supply unit 150 may be configured to be ON/OFF controlled through a user's operation. Only when the power supply unit 150 is controlled to an ON state through a user's operation, the power supply unit 150 is connected to the second connection terminal 120 . An electrical signal of the connected sensing module 200 may be transmitted to the controller 130 .

6 and 7, the connection module 100 is installed on the periphery of the first connection terminal 110, while the first connection terminal 110 is rotatable by a preset angle angle adjustment unit ( 160) may be further included.

The angle adjusting unit 160 is coupled to the first connection terminal 110 and is installed on the main body 140 and the rotating member 161 configured to rotate together with the first connection terminal 110 and is provided inside the The rotating member 161 may be accommodated and may include a guide member 162 for guiding the rotation of the rotating member 161 .

A plurality of projections 161a spaced apart from each other are provided on the outer peripheral surface of the rotating member 161, and a plurality of grooves 162a corresponding to the plurality of projections 161a are provided on the inner peripheral surface of the guide member 162. can Accordingly, the user can arrange the plurality of second connection terminals 120 in predetermined positions by rotating the main body 140 in a state in which the first connection terminal 110 is coupled to the external device 20 . have.

Referring to FIG. 8 , according to an embodiment of the present invention, the connection module 100 and the sensing module 200 may be detachably coupled to each other through magnetism, and may be electrically connected to each other through surface contact. .

To this end, the connection module 100 may include a first coupling member 170 , and the sensing module 200 may include a second coupling member 270 capable of being coupled to the first coupling member 170 . .

Referring to FIG. 9 , the first coupling member 170 includes a coupling protrusion 171 protruding from one end of the connection module 100 and a first magnetic body 172 accommodated at an end of the coupling protrusion 171 . may include

The coupling protrusion 171 may be formed in a wedge structure in which the size of the outer diameter gradually decreases along the protrusion direction. Through this, when the connection module 100 and the sensing module 200 are coupled, an inclined outer circumferential surface of the coupling protrusion 171 is a coupling groove of the second coupling member 270 provided in the sensing module 200 . Guided to the inner peripheral surface of the (271) can be easily coupled.

The second coupling member 270 is formed concavely on the other end of the sensing module 200 facing one end of the connection module 100 , and the coupling groove 271 is formed inside the coupling groove 271 . It is accommodated in the first magnetic body 172 and may include a second magnetic body 272 capable of being coupled through a magnetic force.

The connection module 100 and the sensing module 200 are primarily coupled through the structural coupling of the coupling protrusion 171 and the coupling groove 271, and the first magnetic body 172 and the second As the magnetic body 272 is secondarily coupled through coupling using the magnetism, they may be stably fixed to each other.

Referring to FIG. 8 , in the sensing module 200 , the magnetic force of the second magnetic body 272 is shielded so that the magnetic force of the second magnetic body 272 does not affect other parts of the sensing module 200 . A shielding structure 280 configured to do so may be provided.

The shielding structure 280 may be provided inside the housing 250 and be provided in a shape surrounding the coupling groove 271 of the second coupling member 270 and the periphery of the second magnetic body 272 . have. The shielding structure 280 may be made of the same material as the housing 250 , and may be made of a material capable of shielding magnetic force.

According to an embodiment of the present invention, the connection module 100 and the sensing module 200 may be electrically connected to each other through surface contact coupling. Referring to FIG. 8 , the second connection terminal 120 of the connection module 100 connected to the connection port 240 of the sensing module 200 is a flat plate type that can be electrically connected to each other through surface contact. It may be provided in the form of a terminal.

When the first coupling member 170 of the connection module 100 and the second coupling member 270 of the sensing module 200 are coupled to each other, the connection port 240 and the second connection terminal 120 ) can be electrically connected to each other just by being interviewed.

This makes it easier to combine and separate the connection module 100 and the sensing module 200, and it is possible to prevent the connection port 240 and the second connection terminal 120 from being damaged even in a detachable type. there will be

An identification film may be attached to the end of the sensing module 200 according to an embodiment of the present invention to confirm whether or not the sensing module 200 is used through the presence or absence of damage. At the end of the sensing module 200, a receiving groove (not shown) capable of engaging with a wedge-shaped pressing protrusion (not shown) provided at the end of the connection module 100 is provided, and at the entrance of the receiving groove, the sensing module ( 200) attached to the end of the identification film in the form of a thin film configured to block the entrance of the receiving groove may be attached.

The identification film may be configured to be damaged by being pressed with a predetermined force by the pressing protrusion of the connection module 100 inserted into the receiving groove when the sensing module 200 and the connection module 100 are coupled. Accordingly, the user can determine whether the sensing module 200 is used by checking whether the identification film provided at the end of the sensing module 200 is damaged. However, the configuration capable of confirming whether the sensing module 200 is used is not necessarily limited thereto, and may be changed and applied in various forms.

Referring to FIG. 10 , the sensing module 200 according to an embodiment of the present invention may include a plurality of sensing units configured to detect a target material from an analysis sample. The plurality of sensing units are electrically connected to the connection port 240 , and may be installed inside the housing 250 .

Each of the plurality of sensing units includes the sensor 210 , and the plurality of sensing units are separated from each other through a partition wall 214 , through the connection port 240 electrically coupleable to the connection module 100 . They can be electrically connected to each other.

The plurality of sensing units may include a first sensing unit 210a and a second sensing unit 210b. The first sensing unit 210a and the second sensing unit 210b may be disposed in spaces independent from each other through the partition wall 214 provided in the housing 250 .

The first sensing unit 210a and the second sensing unit 210b respectively detect a target material from the contacted analyte, and react with the target material to generate an electrical signal, the sensor 210 and the bonding wire It may include the connection pad 230 electrically connected to the sensor 210 through 260 and electrically connected to the connection port 240 through the electric wire 261 .

In addition, the first sensing unit 210a and the second sensing unit 210b may each include the channel 253 , and each channel 253 has the nanowell, microwell or milliwell structure. 220 may be provided.

In addition, in each of the first sensing unit 210a and the second sensing unit 210b, between the connection pad 230 and the connection port 240 , the connection pad 230 and the connection port 240 are provided. A power supply unit 290 for controlling the connection may be further provided.

The power supply unit 290 is electrically connected to the connection pad 230 and the connection port 240 , and may be configured to be ON/OFF controlled through a user's manipulation. Accordingly, only when the power supply unit 290 is controlled to be in the ON state through a user's manipulation, the electrical signal of the sensor 210 connected to the connection pad 230 may be transmitted to the connection port 240 .

The biosensor using the nanowell, microwell, or milliwell structure according to the embodiment of the present invention has been described as including the first sensing unit 210a and the second sensing unit 210b, but is limited thereto. No, two or more sensing units may be provided.

The biosensor using the nanowell, microwell or milliwell structure according to the embodiment of the present invention described above has the following effects.

The biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention is configured to be electrically connected to the external device 20 through a connection terminal provided in the form of a USB or pin, and thus various devices It can be applied to , so it is possible not only to reduce costs, but also to use it easily in real life, so that convenience and usability of the product can be increased.

In addition, the biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention includes a connection module 100 electrically connected to an external device 20 and a connection module 100 . Since it consists of a detachable sensing module 200, it is possible to perform various types of inspections through replacement of the sensing module 200, and the sensing module 200 in a state in which the connection module 100 is coupled to the external device 20 It can only be replaced, so that continuous and rapid inspection can be performed.

In addition, in the biosensor 10 using a nanowell, microwell or milliwell structure according to an embodiment of the present invention, the sensing module 200 and the external device 20 are electrically connected to each other via the connection module 100 . Since it is configured to be connected, the sensing portion is prevented from being damaged by the external device 20 even when the biosensor 10 is coupled to or separated from the external device 20 .

In addition, the shape of the electrode provided on the substrate 211 applied to the biosensor 10 using the nanowell, microwell or milliwell structure according to the embodiment of the present invention is improved, so that a larger number of sensors than in the prior art is improved. Since it is possible to manufacture the 210, the manufacturing cost can be reduced.

Specifically, as the shape of the electrode provided on the substrate 211 is improved, the sensor 210 can be manufactured in a size of 2 x 2 mm. Therefore, the number of sensors 210 that can be manufactured using an existing 8-inch wafer has greatly increased from a low of 1000 to a high of 20,000. Except for the sensor 210, the manufacturing cost can be further reduced by using a low-cost PCB process.

In addition, the biosensor 10 using a milliwell or microwell structure according to an embodiment of the present invention can improve resolution by inserting an analyte sample through the nanowell, microwell, or milliwell structure 220 . there are advantages to

Example 1. ALP (alkaline phosphatase) quantitative analysis using nanowell, microwell, and milliwell electrodes

The effectiveness of ALP quantitative analysis using nanowell, microwell, and milliwell electrodes was confirmed.

Specifically, as electrodes, nanowell NWA_U101 electrode (manufactured by Marananotek Co., Ltd.), microwell MWA_U201 (manufactured by Marananotek Co., Ltd.), and Milliwell Milli_U301 (manufactured by Marananotek Co., Ltd.) were used as electrodes. ALP standard solution was prepared as shown in Table 1, and the CUT-OFF value of ALP was formed at 1-3 ng/ml. In this experiment, the concentrations of the positive control and the negative control were prepared as shown in Table 2. The negative control group did not receive ALP. ALP was purchased from randd system, and sigmafast p-nitrophenyl phosphate, a substrate reaction solution, was purchased from sigma. ALP was measured using a 1030C Multi-potentiostat (purchased from CH instrument), and the specific conditions are shown in Table 3.

standard sample 30 ul per test SIGMAFAST 90 ul ALP 10 ul Total 100 ul

division density volume Additive Substrate Solution positive control 3 ng/ml 10 ul sigmafast tablet phosphate (90 ul) 10 ng/ml 30 ng/ml 100 ng/ml 200 ng/ml negative control 0

equipment parameter Detail CH 1030C How to measure SWV (SQUARE WAVE VOLTAMMETRY) start voltage -0.3 V final voltage 0.7 V stair voltage 0.005 V size 0.025 V frequency 10 Hz Setting Sensitivity 1 uA

Samples treated for each concentration as described above were dropped on the prepared electrode, and electrical signals were measured under the conditions shown in Table 3 after 10 minutes. All measurements were repeated three times. Based on the value of the measured current, it was shown as an electrochemical measurement curve graph. As a result, current values measured for each concentration of ALP using the nanowell or microwell electrode were shown in FIGS. 11 and 12 . As the ALP concentration increased, the current value between 0.1 and 0.5 V increased. Among them, the current value increased to the maximum at 0.4 V, so it was easy to detect the ALP concentration, and the measured values for each concentration were recorded at 0.4 V. The current values measured for each concentration of ALP using the milliwell electrode are shown in FIG. 13 . As the ALP concentration increased, the current value between 0.1 and 0.7 V increased. Among them, the current value increased to the maximum at 0.5 to 0.6 V, making it easy to detect the ALP concentration, so the measured values for each concentration were recorded at 0.5 V.

11 is a graph showing current values measured for each concentration of ALP using a nanowell electrode.

12 is a graph showing current values measured for each concentration of ALP using a microwell electrode.

13 is a graph showing current values measured for each concentration of ALP using a milliwell electrode.

In order to confirm the sensitivity of each electrode, the current value measured for each ALP concentration was converted into a ratio obtained by dividing the negative control of each electrode, and it is shown in a graph as shown in FIG. 14 . As a result, the microwell electrode and the nanowell electrode showed a higher current value/negative control value than the milliwell electrode, and the nanowell electrode showed a higher value than the microwell electrode at a high ALP concentration. These results mean that ALP analysis with higher sensitivity than milliwell electrodes is possible when microwell electrodes or nanowell electrodes are used.

14 is a graph showing the conversion of the current value measured for each ALP concentration by the ratio divided by the negative control of each electrode in order to compare the sensitivity of each electrode.

Using the above results, the relationship between the current and the ALP concentration was derived as a function for each electrode as shown in FIGS. 15 to 17 . The function for the current value and ALP concentration at the nanowell electrode can be expressed as y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL)), and the current value and ALP concentration at the microwell electrode The function for can be expressed as y(uA) = -1.35 + 1.15*exp(-1.74*10 ⁷ x(g/mL)), and the function for the current value and ALP concentration at the milliwell electrode is y(uA) ) = -2.76 + 1.63*exp(-3.53*10 ⁷ x(g/mL)). Using this function for each electrode, the ALP concentration can be quantified according to the current value.

15 is a graph showing the relationship between the current value and the ALP concentration in the nanowell electrode.

16 is a graph showing a relationship between a current value and an ALP concentration in a microwell electrode.

17 is a graph illustrating a relationship between a current value and an ALP concentration in a milliwell electrode.

As such, the present invention has been described with reference to the embodiments shown in the drawings, which are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. . Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

10...biosensor 20...external device
100...connection module 110...first connection terminal
120...Second connection terminal 130...Controller
140...Main unit 150...Power supply
160...Angle adjustment unit 161...Rotating unit
162...Guide member 170...First coupling member
171...bonding protrusion 172...first magnetic body
200...sensing module 210...sensor
211...substrate 212...electrode
212a...first electrode 212b...second electrode
212c...third electrode 213...connection pad
214...Bulkhead 220...Milliwell or microwell construction
230...connection pad 240...connection port
250...housing 250a...accommodating space
251... First member 251a... Combination groove
252...Second member 252a...Joining projection
252b...Receiving groove 252c...Collection groove
252d...Sample guide plane 253...Channel
254...Slope 260...bonding wire
261...Wire 270...Second coupling member
271...Coupling groove 272...Second magnetic body
280...shielding structure 290...power

Claims (9)

a connection module electrically connectable to an external device;
A sensing module configured to detect an alkaline phosphatase (ALP) material from the analysis sample introduced into the inside, and transmit an electrical signal generated by reacting with the detected ALP material to the connection module;
The sensing module is provided with a sensor that detects the ALP material from the analyte sample, reacts with the ALP material to generate the electrical signal, and quantitatively measures the ALP material by the generated electrical signal,
The sensor is
and a plurality of electrodes provided on one surface of the substrate and reacting with the ALP material to generate an electrochemical signal,
The sensing module is a biosensor for ALP measurement that is detachable from the connection module. The biosensor for measuring ALP according to claim 1, wherein the sensor comprises a nanowell, microwell, or milliwell structure having a plurality of grooves on the electrode. The biosensor for measuring ALP according to claim 2, wherein the nanowell, microwell or milliwell has a diameter of 50 nm to 50 mm. The biosensor of claim 1, wherein the analysis sample is a biological sample isolated from an individual. The biosensor of claim 1, wherein the electrode is a working electrode, a counter electrode, or a reference electrode. The biosensor for measuring ALP according to claim 1, wherein the connection module and the sensing module are detachably coupled to each other through magnetism, and electrically connected to each other through surface contact. The biosensor for measuring ALP according to claim 1, wherein the sensing module is at least one, and the at least one sensing module is detachable from the one connection module. The method according to claim 1, wherein the sensing module is provided with a plurality of sensing units including the sensor, the plurality of sensing units are separated from each other through a barrier rib, and are electrically connected to each other through a connection port electrically coupleable to the connection module. A biosensor for ALP measurement, characterized in that. The biosensor for measuring ALP according to claim 1, wherein the quantitative measurement of the ALP material is measured by any one of Equations 1 to 3.
[Equation 1]
y(uA) = -257.00 + 256.96*exp(-6304.58x(g/mL))
[Equation 2]
y(uA) = -1.35 + 1.15*exp(-1.74*107x(g/mL))
[Equation 3]
y(uA) = -2.76 + 1.63*exp(-3.53*107x(g/mL))

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